Current Pharmaceutical Analysis - Volume 16, Issue 8, 2020

Background: Schizophrenia is a severe mental illness that affects more than twenty-one million people throughout the world. Schizophrenia also causes early death. Schizophrenia and other related psychotic ailments are controlled by the prescription of antipsychotic drugs, which act by blocking certain chemical receptors in the brain and thus relieves the symptoms of psychotic disorder. These drugs are present in the different dosage forms in the market and provided in a certain amount as per the need of the patients. Objective: Since such medications treat mental disorders, it is very important to have a perfect and accurate dose so that the risk factor is not affected by a higher or lower dose, which is not sufficient for the treatment. For accurate assay of these kinds of drugs, different analytical methods were developed ranging from older spectrophotometric techniques to latest hyphenated methods. Results: The current review highlights the role of different analytical techniques that were employed in the determination and identification of antipsychotic drugs and their metabolites. Techniques such as spectrophotometry, fluorimetry, liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry employed in the method development of such antipsychotic drugs were reported in the review. Different metabolites, identified using the hyphenated techniques, were also mentioned in the review. The synthesis pathways of few of the metabolites were mentioned. Conclusion: The review summarizes the analyses of different antipsychotic drugs and their metabolites. A brief introduction of illnesses and their symptoms and possible medications were highlighted. Synthesis pathways of the associated metabolites were also mentioned.

Pharmaceutical analysis plays an indispensable role in various stages of drug pipeline including drug development, fabrication of formulation, stability studies and quality control. It is also used for characterizing the composition of different dosage forms in quantitative and qualitative ways. Comprehensive literature survey forms the foundation stone for the focused analysis of research activity. Irbesartan is a hypertension antagonist chiefly employed in the treatment of high blood pressure which is an Angiotensin II Receptor blocker. The multiple mixtures of various antihypertensive medicaments raise challenges in the method development and validation. A genuine venture is undertaken to compile the literatures related to the antihypertensive drug Irbesartan published in various peer reviewed journals. HPLC and UV spectrophotometry are the most preferred analytical techniques when compared to other methods. This present review provides an in-depth assortment of various analytical techniques published for Irbesartan and its combinations, which will help the researchers in their future endeavors.

Background: Duloxetine hydrochloride (DUL) is a serotonin-norepinephrine reuptake inhibitor. It is used for treating depression and anxiety. It is available in the market as a capsule called Cymbatex®, which is used for the treatment of depression. 1-naphthol is a potential impurity of DUL. It is hepatotoxic to humans and has potential toxicity to freshwater fish. Objective: Duloxetine hydrochloride was determined in the presence of its toxic impurity 1-naphthol in raw material and in pharmaceutical dosage forms using three multivariate calibration chemometric methods. Methods: Classical Least Squares (CLS), Partial Least-Squares (PLS) and linear support vector regression (SVR) were developed using UV spectral data. The three methods were compared among each other and the advantages and disadvantages were discussed. For good results, a two-factor four-level experimental design was used, resulting in a training set of 16 mixtures containing different ratios of each component. The test set consisting of nine mixtures was necessary to test the ability of the proposed methods to predict DUL in the presence of its impurity, 1-naphthol. Results: The results show the success of the three developed methods to determine DUL in the presence of small levels of its toxic impurity with good accuracy and selectivity. The results of the dosage form were compared statistically to that of the reported HPLC method, with no significant difference in accuracy and precision. Conclusion: The suggested calibration models are suitable for routine analysis of the drug in bulk and pharmaceutical dosage forms. Compared to the CLS and PLS models, the SVR model gives the best results regarding the accuracy with a lower prediction error and better generalization ability. However, the CLS and PLS models are found to be simpler and faster in usage and management.

Background: Atenolol is a selective beta 1 blocker that can be used alone or in combination with hydrochlorothiazide or with chlorthalidone for the treatment of hypertension and prevention from a heart attack. Objective: The main target of this work was to improve modern, easy, accurate and selective liquid chromatographic method (RP-HPLC) for the determination of these drugs in the presence of their degradation products. These methods can be used as analytical gadgets in quality control laboratories for a routine examination. Methods: In this method, the separation was accomplished through an Inertsil® ODS-3V C18 column (250 mm x 4.6 mm, 5 μm), the mobile phase used was 25 mM aqueous potassium dihydrogen orthophosphate solution adjusted to pH 6.8 by using 0.1M sodium hydroxide and acetonitrile (77 : 23, v/v), the flow rate used was 1 ml/min and detection was achieved at 235 nm using UV. Results: All peaks were sharp and well separated, the retention times were atenolol degradation (ATN Deg.) 2.311 min, atenolol (ATN) 2.580 min, hydrochlorothiazide degradation (HCT Deg.) 5.890 min, hydrochlorothiazide (HCT) 7.016 min, chlorthalidone degradation CTD Deg 8.018 min and chlorthalidone (CTD) 14.972 min. Linearity was obtained and the range of concentrations was 20- 160 μg/ml for atenolol, 10-80 μg/ml for hydrochlorothiazide and 10-80 μg/ml for chlorthalidone. According to ICH guidelines, method validation was accomplished, these methods include linearity, accuracy, selectivity, precision and robustness. Conclusion: The optimized method demonstrated to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical dosage forms.

Background: Curcumin (CUR) and baicalein (BAI) are the main active ingredients in Curcuma longa and Scutellaria baicalensis, which are used together in Jiang-Qin-Si-Wu decoction to treat gynecological diseases. On the other hand, zebrafish, as a metabolic model has become more popular, therefore, the metabolism of CUR and BAI in zebrafish is investigated in the present study. Methods: Zebrafish embryos after hatching 48 hours were divided into four experimental groups. The blank group was exposed to 1 mL of ultra-pure water. Three drug-treated groups were exposed to CUR (8 μM, 1 mL), BAI (8 μM, 1 mL), CUR and BAI (8 μM, 2 mL), respectively. After homogenization, they were analyzed by liquid chromatography-tandem mass spectrometry (LCMS/ MS). The structure of the metabolites was determined by comparing their corresponding mass spectra with those of relevant literature. According to the change of metabolite content, the metabolic effect of curcumin and baicalein was explored. Results: Five and six metabolites of CUR and BAI in zebrafish were identified by LC-MS/MS, respectively. Their metabolic pathways in zebrafish were glucuronidation and sulfation. Reduction and methylation reactions also occurred for CUR and BAI, respectively. In addition, after combined exposure of both the drugs, CUR reduced the BAI glucuronide metabolites and inhibited the metabolism of BAI in zebrafish, which is consistent with the mammalian metabolism. Conclusion: Using LC-MS/MS analysis, zebrafish is a feasible model for drug metabolism study. The results of metabolic study indicated that CUR might affect the therapeutic effect of BAI.

Background: Linezolid is a synthetic broad-spectrum antibacterial belonging to the class of oxazolidinones. Linezolid for intravenous infusion is isotonized with dextrose. In acidic environment, the dehydration of dextrose produces furan derivatives, 5-hydroxymethylfurfural (5-HMF) being the main one. The determination of this degradation product is of fundamental importance, since there is evidence it is cytotoxic, genotoxic, mutagenic and carcinogenic. However, there is no official method for the determination of 5-HMF in drug products. Objective: The aim of this study was to develop and validate a high performance liquid chromatographic method to quantify 5-HMF in injection of linezolid. Methods: The chromatographic separation, after optimization, was performed on C18 (150 x 4.6 mm, 5 μm) column. Mobile phase was composed of 14 mM potassium phosphate buffer pH 3.0 ([H⁺] = 1.0 x 10^-3) and methanol in gradient elution at 1.0 mL min^-1. The injection volume was 10 μL and detection was performed at 285 nm. Results: The method was optimized and validated, showing selectivity, linearity in the range from 0.075 to 9.0 μg mL^-1, precision (RSD ≤ 2.0%), accuracy (mean recovery of 100.07%) and robustness for temperature and pH variation. Conclusion: The method was shown to be adequate to determine 5-HMF in injection containing linezolid in routine analysis.

Background: Tamoxifen (Soltamox) is an antineoplastic agent and an estrogen receptor antagonist used to treat breast cancer, but have severe side effects such as hot flashes, vaginal discharge, etc. Dose monitoring is a necessity for optimum treatment, to prevent severe adverse effects. Methods: In this study, the solvent bar microextraction method (SBME) was used for preconcentration and microextraction coupled with High-performance liquid chromatography-ultraviolet (HPLC-UV) analysis of tamoxifen. Results: The limit of detection and limit of quantification were 13.3 and 40 μgL-1, respectively. The linear range was between 40 and 10000 μgL-1 with a correlation coefficient of 0.999. The enrichment factor was 169 and the relative standard deviation within-day and between-day were 3.6 and 4.0, respectively. Conclusion: The use of sensitive SBME method coupled with HPLC-UV analysis for detection of tamoxifen at trace level proved to be successful, offering a desirable preconcentration factor, and a costeffective and green set-up for determining its rate of elimination from cancer patients and wastewater.

Introduction: Saxagliptin hydrochloride is an oral hypoglycemic agent used for the treatment of type 2 diabetes mellitus. Saxagliptin is unstable because it undergoes an intra-molecular cyclisation reaction to form a cyclicamidine in both solution and solid states. In pharmaceutical development of saxagliptin it is important to select the excipients which are compatible and help to minimize the formation of cyclicamidine. In excipient compatibility study for saxagliptin it is essential to identify the formation of cyclicamidine and other related substances. Materials and Methods: In the current work, the method for quantification of saxagliptin, cyclicamidine and its related substances by high performance liquid chromatographic was developed and validated. This method was used as screening technique for assessing the compatibility of saxagliptin with some pharmaceutical excipients. These were evaluated by analyzing the pure saxagliptin and saxagliptin- excipient in physical mixture, which were stored under different conditions at 40°C/75% Relative Humidity (RH) for one month. The method was successfully validated as per ICH guidelines. Results and Conclusion: The results of compatibility study demonstrate the suitability of saxagliptin with Methocel, Polyethylene Glycol (PEG), Opadry Red, Opadry pink, Opadry white, and Opadry Pink.

Background: Simultaneous determination of medication components in pharmaceutical samples using ordinary methods have some difficulties and therefore these determinations usually were made by expensive methods and instruments. Chemometric methods are an effective way to analyze several components simultaneously. Objective: In this paper, a novel approach based on Bayesian regularized artificial neural network is developed for the determination of Loratadine, Naproxen, and Diclofenac in water using UV-Vis spectroscopy. Methods: A dataset is collected by performing several chemical experiments and recording the UV-Vis spectra and actual constituent values. The effect of a different number of neurons in the hidden layer was analyzed based on final mean square error, and the optimum number was selected. Principle Component Analysis (PCA) was also applied to the data. Other back-propagation methods, such as Levenberg-Marquardt, scaled conjugate gradient, and resilient backpropagation, were tested. Results: In order to see the proposed network performance, it was performed on two crossvalidation methods, namely partitioning data into train and test parts, and leave-one-out technique. Mean square errors between expected results and predicted ones implied that the proposed method has a strong ability in predicting the expected values. Conclusion: The results showed that the Bayesian regularization algorithm has the best performance among other methods for simultaneous determination of Loratadine, Naproxen, and Diclofenac in water samples.

Background: Commelina communis Linn (Commelinae Herba) is a traditional Chinese medicine that can be used both as food and as medicine. It has been used to treat a variety of disorders, including a cold, high fever, sore throat, edema and oliguria for many years. Two pairs of isomeric flavonoid glycosides are the main active components in Commelina communis Linn, and they have a high content. Objective: The objective of this study was to determine the pharmacodynamic and pharmacological effects of Commelina communis Linn. Method: A sensitive, efficient, and rapid LC-MS/MS method was developed to simultaneously identify two pairs of isomeric flavonoid glycosides in rats. Chromatographic separation was carried out on a Wonda Cract ODS-2 C18 column (150 mm x 4.6 mm, 5 μm) using a mobile phase composed of 0.1% formic acid (aqueous solution) and methanol at a flow rate of 0.8 mL/min. The detection of the four analytes and the internal standard (IS) sulfamethoxazole was performed with multiple reaction monitoring (MRM) in negative electrospray ionization mode. All the analytes were eluted within 20 min. Results: This method was successfully applied for simultaneous identification of the concentrations of the four compounds in the plasma after the oral administration of 10 mL/kg Commelina communis Linn extract to rats. The pharmacokinetic study indicated that analytes reached their Cmax in approximately 15 min and could be detected until 12 h. Conclusion: The method complies with the State Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery and stability. This is the first report on the pharmacokinetics of Commelina communis Linn. The information gained from this research may be valuable for the preclinical and clinical applications of Commelina communis Linn.

Background: A simple and sensitive ultra-performance liquid chromatography-mass spectrometry method was developed and validated to measure the concentrations of gemigliptin (GEM) and teneligliptin (TEN) using pioglitazone (PIO) as an internal standard. Methods: Chromatographic separation of two gliptins was achieved on a C-18 (100 mm X 2.1 mm, 2.7 μm) column using a mobile phase consisting of formic acid in water (0.1 % v/ v): acetonitrile in gradient elution. Electrospray ionization (ESI) source was operated in positive mode (ionization). Targeted MS-MS mode on a quadrupole time of flight (Q-TOF) mass spectrometer was used to quantify the drugs utilizing the mass transitions of 490.1 (m/z), 427.2 (m/z) and 357.1 (m/z) for GEM, TEN and PIO, respectively. Results: As per ICH Q2R1 guidelines, a detailed validation of the method was carried out and the standard curves were found to be linear between the concentration ranges of 509.8-1529.4 ng mL-1 and 510.6-1531.7 ng mL-1 for GEM and TEN, respectively. Precision and accuracy results were found to be within the acceptable limits. The mean recovery was found to be 98.8± 0.76 % (GEM) and 98.6 ±0.98 % (TEN), respectively. Conclusion: The optimized validated UPLC-QTOF (MS-MS) method offered the advantage of shorter analytical times and higher sensitivity and selectivity to the nanogram level.

Background: Somatropin is recombinant human growth (GH) used for the treatment of growth failure in children and GH deficiency in adults. At present, rhGH marketed in China is mostly freeze-dried powder injection. As the lyophilization process is unstable, time-consuming and costly, rhGH has been prepared into an aqueous solution for administering directly. Introduction: In this study, the pharmacokinetics of two dosage forms of rhGH in beagle dogs after single subcutaneous administration was determined by enzyme-linked immunosorbent assay (ELISA). Methods: Twelve healthy beagles (male, 6:female, 6) were used for the pharmacokinetic study and were equally divided into two groups. Subcutaneous injection of 0.2 IU/kg with rhGH in the two formulations. The blood samples were taken from forearms, 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 3, 4, 7, 10, 24 h and collected the beagle plasma on time. The pharmacokinetic parameters of rhGH after subcutaneous (s.c.) injection were determined experimentally on beagles. Primary PK endpoints were area under the serum concentration-time curve (AUC0-t) and maximum serum concentration (Cmax). Serum rhGH level was determined by enzyme-linked immunosorbent assay. Results: The calibration curves obtained were linear over the concentration range of 25 to 1600 ng/ml for recombinant human growth. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. The analysis samples were stable under different storage conditions and temperature. Conclusion: The developed ELISA method has been successfully applied to the studies of pharmacokinetic of recombinant human growth hormone in beagles.

Background: Because of the similar appearance of Gynura segetum and panax notoginseng, the patients often mistakenly use Gynura segetum as Panax notoginseng, which causes serious liver damage. There is no comparative study on the metabolism of Gynura segetum and Panax notoginseng in the literature. This study was conducted to compare the difference between Panax notoginseng and its counterfeit Gynura segetum by using metabolomics method. Methods: In this paper, an ultra performance liquid chromatography coupled to quadrupole time-offlight mass spectrometric(UPLC-Q/TOF/MS) were used to detect the type of endogenous metabolites in urine and plasma of three groups (normal group, ethanol extract of panax notoginseng, decoction of Gynura segetum respectively, and different multivariate statistical analysis methods were established. Results: In this experiment, main urine biomarkers were L-glutamate, L-methionine, cytidine, and Ltyrosine in the Panax notoginseng group, which are phytosphingosine, creatine and sphinganine in the Gynura segetum group. The plasma biomarkers identified in the Panax notoginseng group were arachidonic acid, L-tyrosine, linoleic acid, alpha-linolenoyl ethanolamide and lysoPC (15:0), and in the Gynura segetum group are L-arginine, L-valine, arachidonic acid and LysoPC(18:2(9Z,12Z)). Conclusion: There are significant difference between Panax notoginseng and Gynura segetum in biomarkers from the perspective of metabolomics in the body.

Background: Regadenoson is an A2A adenosine receptor agonist that is a coronary vasodilator and commonly used as a pharmacologic cardiac stressing agents. Methods: HPLC method was used for the analysis of related substances. The degraded impurities during the process were isolated and characterized by IR, Mass and NMR spectral analysis. Results: Forced degradation study of regadenoson under conditions of hydrolysis (neutral, acidic and alkaline) and oxidations suggested in the ICH Q1A(R2) was accomplished. The drug showed significant degradation under all the above conditions. On the whole, five novel degradation products were found under diverse conditions along with process related impurities which were not reported earlier. Conclusion: All the degradation products were well characterized by using advanced spectroscopic techniques like IR, 1H NMR, 13C NMR and Mass spectra. The identification of these impurities will be productive for the quality control during the production and stability behavior of the regadenoson drug substance.

Background: Vorasidenib is a pan-IDH inhibitor, undergoing clinical trials for the treatment of acute myeloid leukemia. Methods: In this paper, we present the data of method validation to quantify vorasidenib in the mice blood mice using dried blood spot (DBS) method on LC-MS/MS as per FDA bioanalytical method validation guideline. Using methanol (enriched with internal standard) as an extraction solvent followed by sonication, vorasidenib was extracted from DBS quality control samples, calibration curve samples and pharmacokinetic study samples. Baseline separation of vorasidenib and the IS in a 2.0 μL injected sample was accomplished by delivering 0.2% formic acid and acetonitrile (25:75, v/v) at a constant flowrate (1.00 mL/min) on a C18 column. The total run time was 2.0 min. Using the transition pair of m/z 415.4→260.4 for vorasidenib and m/z 583.1→186.1 for the IS, the quantitation was performed. The method linearity range was 1.00-3008.00 ng/mL. Results: The recovery of vorasidenib ranged between 71.28%-78.14% across the tested concentrations. No matrix effect was seen. Intra- and inter-day precisions were ≤7.23% and intra- and inter-accuracies ranged between 97.1%-107%. Vorasidenib was stable for three freeze/thaw cycles, up to 7 days at room temperature and for one month at -80°C. Following intravenous and oral administration of vorasidenib to mice, it was quantifiable up to 72 h. The oral bioavailability was 51.6%. Conclusion: All the validation parameters met the acceptance criteria as specified in the FDA regulatory guideline. The results suggest that validated DBS method can be used for pharmacokinetic studies in mice to characterize the pharmacokinetic parameters of vorasidenib post intravenous and oral administration.

Objective: A rapid and environmental friendly microemulsion liquid chromatographic method was developed for simultaneous quantification of morphine (MOR), tramadol (TRA) and lornoxicam (LOR) in biological fluids. Methods: Microemulsion used in this study was an aqueous solution containing sodium dodecyl sulfate (0.12 M), n-propanol (10%), ethyl acetate (0.75%), tri-ethyl amine (0.3%), orthophosphoric acid (0.15 %) and the pH was adjusted to 3.0 with orthophosphoric acid. Chromatographic separation was carried out on a monolithic C18 column and a mobile phase flow rate of 1.0 mL min−1 was applied throughout the analysis. The data was monitored using UV-detection mode at a wavelength of 220 nm. Results: Under the optimized conditions, all the studied drugs were well resolved and completely eluted within 6 min. The proposed method was linear over the concentration ranges of 0.5−100, 0.75−125 and 0.25−50 μg mL−1; limits of detection of 0.074, 0.086 and 0.056 μgmL−1 and limits of quantification of 0.21, 0.28 and 0.12 were recorded for TRA, LOR, and MOR, respectively. The developed method was fully validated according to the ICH guidelines. The method was successfully utilized to estimate the tested analytes in plasma and urine samples, which were directly injected into the chromatographic system after suitable dilution with the microemulsion. Conclusion: The developed method is considered to be very efficient to analyze the cited drugs in different biological fluids with low running costs and short analysis time.